[0004]Dopamine is a chemical that the body produces naturally in the
brain. In the brain, dopamine functions as a neurotransmitter and is a
critical component used by the brain to control bodily movements,
therefore changes in the level of dopamine in the brain can have
devastating results. Augmentation in the levels of dopamine in the brain
has been associated with conditions such as drug addition, psychiatric
disorders like schizophrenia, depression, Parkinson's Disease, and
Parkinsonian-like disorders.

[0005]Parkinson's Disease (PD) is a progressive disorder of the central
nervous system that affects over one million people in the United States
alone and is associated with a loss in dopamine production in a specific
area of the brain. Approximately 40,000 Americans are diagnosed with PD
every year, and although PD is typically equated to be a disease
afflicting older adults, more and more people are being diagnosed with
the disease before the reach the age of 50. It is a chronic and
progressive disease, meaning that the symptoms of PD grow worse and last
over time. Characteristic symptoms include a decrease in spontaneous
movements, gait difficulty, postural instability, rigidity and tremors.

[0006]As with a number of neurological diseases, the true cause of PD is
not known. However, research has shown that Parkinson's Disease occurs
when a group of neuronal cells in the area of the brain called the
substantia nigra pars compacta (SNc), begin to malfunction and eventually
die leading to a decrease in levels of dopamine in the brain, which in
turn leads to impaired motor control and coordination. There is currently
no known cure for PD. To date, treatment has been directed to increasing
the amount of dopamine in the brain by drug administration, or to more
invasive surgical treatments such as targeted neuronal ablation and deep
brain stimulation. Unfortunately, all treatments suffer from drawbacks,
some serious, which debilitate the patient and compromise the quality of
life.

[0007]What are needed are novel ways of understanding and studying these
types of dopamine related disorders and diseases, and novel ways of
treating these types of disorders and diseases without disrupting normal
neuronal functioning and compromising the quality of life of those
afflicted.

[0009]The neurodegenerative disorder Parkinson's Disease is caused by the
death of dopaminergic neurons in the substantia nigra pars compacta
(SNc). The etiology of the disease is not understood, however mutations
in genes associated with mitochondria or protein folding are associated
with human PD. These genes, however, are not expressed exclusively in the
dopaminergic SNc neurons and animals harboring these mutations do not
necessarily develop, nor show signs, of the disease. The majority of PD
cases are not associated with any known genetic defect, and most of the
current thinking suggests that exposure to environmental toxins, like
rotenone, are responsible for these cases. Rotenone does selectively kill
SNc DA neurons in rats, but it is not clear why as it is a mitochondrial
toxin that enters and affects all neurons. Other dopaminergic disorders
include mental disorders (e.g., schizophrenia, depression, drug
addiction) and Parkinsonian-like disorders (e.g., juvenile parkinsonism,
Ramsey-Hunt paralysis syndrome).

[0010]Most medications used to treat Parkinson's Disease and other
dopaminergic system disorders, either mimic the effect of dopamine,
increase dopamine levels, or extend the action of dopamine in the brain.
The gold standard by which all treatments for PD are measured is the
administration of levodopa, which is a substance that is converted into
dopamine in the brain. However, levodopa typically is administered as
part of a chemical cocktail, for example with carbidopa that prevents the
levodopa from being converted to dopamine in the bloodstream, and/or
entacapone which extends the time levodopa is active in the brain. There
are also a number of dopamine agonists which are administered, e.g.,
bromocriptine, pergolide, pramipexole, and ropinirole. Additional
treatments include chemicals that do not act directly on the dopaminergic
system, but alternatively target another neurotransmitter, acetylcholine,
which is in overabundance when the dopaminergic system ceases to create
dopamine. The imbalance of acetylcholine relative to dopamine levels
causes additional physiological problems. Those chemicals that target
acetylcholine, anticholinergics, include trihexypheidyl, benzotropine
mesylate, MAO and COMT inhibitors such as selegiline, deprenyl,
entacapone (previously described), and tolcapone are sometimes
administered to help prolong the efficacy of the levodopa by slowing its
breakdown in the brain, thereby helping to provide a more stable,
constant supply of levodopa. Unfortunately, all of these drugs alone or
in combination are associated with a variety of side effects, and
potential drug interaction problems, which make their usage less than
desirable. Other extreme, more invasive, measures to correct dopaminergic
disorders include the targeted destruction of afflicted neuronal cells in
the affected area of the brain, and deep brain stimulus. Both of these
invasive procedures carry the risk of stroke and other operative
complications.

[0011]It is contemplated that the death of dopaminergic neurons can be
attributed to their reliance upon calcium channels. The calcium channels
found in dopaminergic neurons, voltage-gated L-type calcium channels of
which Cav1.3a is one, autonomously generate influxes of calcium into
dopaminergic neurons. The intracellular calcium loading caused by this
process, also referred to as pacemaking, synergizes with the stress
created by the factors that potentially cause dopaminergic disorders
(e.g., environmental toxins, genetic mutations, and the like) thereby
inducing preferential death of the dopaminergic neurons and the onset of
disease. Young `juvenile` neurons don't depend on calcium to the extent
that aged neurons do, i.e. as neurons age they require more calcium. It
is contemplated that by reducing the calcium loading during pacemaking,
the neurons will be forced to convert to a more `juvenile` form of
pacemaking (an altered pacemaking mechanism), one that relies upon better
tolerated ions such as sodium. This can be achieved by modulating (e.g.,
disrupting) pharmacologically or genetically the Cav1.3a channels in
the SNc dopaminergic neurons.

[0012]Current pharmacological therapies depend upon continued viability of
dopaminergic neurons affected by the diseases. However, as the disease
progresses and these neurons die, current pharmacological approaches fail
to provide symptomatic relief. None of the current therapies slow
progression of the disease or the death or dopaminergic neurons. By
directly targeting the Cav1.3a channels as described in the present
invention, damaged cells are protected from further degradation and
eventual death. Additionally, pre-treatment of subjects, by targeting
Cav1.3a channels using the methods and compositions of the present
invention, who may be pre-disposed to developing a dopaminergic disorder
(e.g., via environmental toxin exposure, genetic disposition) provides a
preventative therapy for these types of dopaminergic disorders.

[0013]There is a real need for novel ways to treat patients with
dopaminergic disorders which bypass the dopaminergic system thereby
allowing an alternative treatment for those afflicted with these types of
disorders. It is contemplated that alternative treatments would bypass
the side effects associated with present treatment regimes. Therefore,
the present invention relates to methods and compositions for modulating
calcium channels. In particular, the present invention provides methods,
compositions, and kits for modulating (e.g., disrupting) Cav1.3a
calcium channels. The methods, compositions, and kits described herein
can be utilized to provide novel ways of treating and studying
dopaminergic related disorders.

[0014]In one embodiment, the method of the present invention is a method
of treatment for dopaminergic disorders comprising administering a
compound that inhibits a voltage-gated calcium channel of the type
Cav1.3a to a subject having a dopaminergic disorder. In some
embodiments, the method comprises the administration of a calcium channel
blocker, preferably a dihydropyridine calcium channel blocker. In some
embodiments, the dihydropyridine calcium channel blocker comprises
nifedipine, nimodipine, and/or isradipine.

[0015]In one embodiment, the method of the present invention is a method
to identify compounds that inhibit activity and/or expression of a
voltage-gated calcium channel of the type Cav1.3a comprising
providing a compound suspected of inhibiting the expression or activity
of a Cav1.3a calcium channel, applying the compound to a sample
which contains Cav1.3a calcium channels, and determining whether or
not the compound has affected the activity and/or expression of
Cav1.3a calcium channels. In some embodiments, the compounds to be
tested comprise nucleic acids (e.g., siRNA), and small molecules,
antibodies, peptides, proteins, and the like.

[0016]In one embodiment, the method of the present invention is a method
of co-therapy treatment for dopaminergic disorders comprising providing a
compound that inhibits the activity and/or expression of a voltage-gated
calcium channel of the type Cav1.3a in conjunction with an
additional therapeutic compound that is useful in treating dopaminergic
disorders, and administering the combination to a subject suspected of
having a dopaminergic disorder. In some embodiments, the additional
therapeutic compound comprises a dihydropyridine calcium channel blocker
and/or a nucleic acid. In some embodiments, the additional therapeutic
agent comprises levodopa, carbidopa, entacapone, apomorphine
hydrochloride, bromocriptine, pergolide, pramipexole, ropinirole,
benzotropine mesylate, trihexyphenidyl HCl), selegiline, tolcapone,
amantadine, riluzole, and/or L-dopa ethyl ether.

[0017]In one embodiment, the composition of the present invention
comprises a compound that inhibits the activity and/or expression of
voltage-gated calcium channels of the type Cav1.3a and is useful in
treating dopaminergic disorders. In some embodiments, the compound that
inhibits a Cav1.3a calcium channel comprises a calcium channel
blocker and/or a nucleic acid. In some embodiments, the calcium channel
blocker comprises a dihydropyridine calcium channel blocker. In some
embodiments, the dihydropyridine calcium channel blocker comprises
nifedipine, nimodipine, and/or isradipine. In some embodiments, a
compound that inhibits the activity and/or expression of voltage-gated
calcium channel of the type Cav1.3a is a nucleic acid, preferably a
small interfering RNA. In some embodiments, the compound that inhibits
the activity and/or expression of voltage-gated calcium channel of the
type Cav1.3a is further combined with an additional therapeutic
agent comprising levodopa, carbidopa, entacapone, apomorphine
hydrochloride, bromocriptine, pergolide, pramipexole, ropinirole,
benzotropine mesylate, trihexyphenidyl HCl), selegiline, tolcapone,
amantadine, riluzole, and/or L-dopa ethyl ether.

DESCRIPTION OF THE FIGURES

[0018]FIG. 1 shows inhibition of L-type calcium channels with the
dihydropyridine calcium channel antagonist nimodipine, which inhibits
autonomous pacemaking of dopaminergic neurons in a tissue slice from an
adult.

[0020]FIG. 3 demonstrates that pacemaking is accompanied by large
fluctuations in the concentration of dendritic calcium in SNc
dopaminergic neurons.

[0021]FIG. 4 shows that the altered pacemaking mechanism can be elicited
in an adult SNc dopaminergic neuron by inhibiting Cav1.3 channels
for a brief period. Recordings are shown from a SNc dopaminergic neuron
before, during, and after application with the dihydropyridine calcium
channel blocker isradipine.

[0022]FIG. 5 demonstrates that knocking out Cav1.3 channels confers
resistance to the pesticide rotenone, a member of a class of
environmental agents thought to contribute to idiopathic Parkinson's
Disease. Sections from wt and Cav1.3 knock-out mouse brains are
stained for tyrosine hydroxylase after application of 100 nM rotenone and
10 μM glibenclamide.

DEFINITIONS

[0023]As used herein, the term "sample" is used in its broadest sense. In
one sense, it is meant to include a specimen or culture obtained from any
source, as well as biological and environmental samples. Biological
samples may be obtained from animals (including humans) and encompass
fluids, solids, tissues, and gases. Biological samples include tissues
and blood products, such as plasma, serum and the like. Such examples are
not however to be construed as limiting the sample types applicable to
the present invention.

[0024]As used herein, the term "peptide" refers to a compound comprising
from two or more amino acid residues wherein the amino group of one amino
acid is linked to the carboxyl group of another amino acid by a peptide
bond. A peptide can be, for example, derived or removed from a native
protein by enzymatic or chemical cleavage, or can be prepared using
conventional peptide synthesis techniques (e.g. solid phase synthesis) or
molecular biology techniques (see Sambrook, J. et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
(1989)).

[0025]As used herein, the term "peptidomimetic" refers to molecules which
are not polypeptides, but which mimic aspects of their structures. For
example, polysaccharides can be prepared that have the same functional
groups as peptides. A peptidomimetic comprises at least two components,
the binding moiety or moieties, and the backbone or supporting structure.

[0026]As used herein, the term "antibody" encompasses both monoclonal and
polyclonal full length antibodies and functional fragments thereof (e.g.
maintenance of binding to target molecule). Antibodies can include those
that are chimeric, humanized, primatized, veneered or single chain
antibodies.

[0027]As used herein, the term "dopaminergic disorder" refers to diseases
and conditions associated with aberrant dopamine production. Dopaminergic
disorders include, but are not limited to, Parkinson's Disease, and
Parkinsonian-like disorders such as juvenile parkinsonism and Ramsey-Hunt
paralysis syndrome.

[0028]As used herein, the term "effective amount" of a therapeutic
compound (e.g. agent, compound, or drug) is an amount sufficient to
achieve a desired therapeutic and/or prophylactic effect, such as to
inhibit neuronal cell death, or to alleviate behavioral disorders
associated with a dopaminergic disorder.

[0029]As used herein, the terms "agent", "compound" or "drug" are used to
denote a compound or mixture of chemical compounds, a biological
macromolecule such as an antibody, a nucleic acid, or an extract made
from biological materials such as bacteria, plants, fungi, or animal
(particularly mammalian) cells or tissues that are suspected of having
therapeutic properties. The compound, agent or drug may be purified,
substantially purified or partially purified.

[0030]As used herein, the term "fragment" when in reference to a protein
(e.g. "a fragment of a given protein") refers to portions of that
protein. The fragments may range in size from two amino acid residues to
the entire amino acid sequence minus one amino acid. In one embodiment,
the present invention contemplates "functional fragments" of a protein.
Such fragments are "functional" if they can bind with their intended
target protein (e.g. the functional fragment may lack the activity of the
full length protein, but binding between the functional fragment and the
target protein is maintained).

[0031]As used herein, the term "antagonist" refers to molecules or
compounds (either native or synthetic) that inhibit the action of a
compound (e.g., receptor channel, ion channel, etc.). Antagonists may or
may not be homologous to these compounds in respect to conformation,
charge or other characteristics. Thus, antagonists may be recognized by
the same or different receptors that are recognized by an agonist.
Antagonists may have allosteric effects that prevent the action of an
agonist. Or, antagonists may prevent the function of the agonist.

[0032]As used herein, the term "therapeutically effective amount" refers
to that amount of a composition which results in amelioration of symptoms
or a prolongation of survival in a patient. A therapeutically relevant
amount relieves to some extent one or more symptoms of a disease or
condition, or returns to normal, either partially or completely, one or
more physiological or biochemical parameters associated with a disease or
condition.

[0033]As used herein, the term "subject" refers to any biological entity
that can be used for experimental work. For example, a "subject" can be a
mammal such as a mouse, rat, pig, dog, non-human primate. Preferably the
subject is a human primate.

DETAILED DESCRIPTION OF THE INVENTION

[0034]Neurons express multiple types of voltage-gated calcium (Ca2+)
channels (Cav). Neuronal N-type and P/Q type Ca2+ channels have
been shown to mediate Ca2+ influx that triggers release of
neurotransmitters. Neuronal L-type Ca2+ channels do not trigger such
a release even though they still play a critical role in Ca2+ influx
in neurons. There are two distinctive subtypes of neuronal L-type
Ca2+ channels; Cav 1.2 and Cav 1.3. The Cav 1.3
channel subtype is further alternatively spiced at its C-terminus to
yield two forms of Cav 1.3; Cav 1.3a (long splice variant) and
Cav 1.3b (short splice variant). The long splice variant Cav
1.3a contains SH3 and class I PDZ binding domains that selectively bind
to Shank scaffolding proteins, the combination of which has been found
targeted to glutamatergic synapses in striatal medium spiny neurons
(MSN). This scaffolding interaction enables modulation of the channel by
the two dopamine receptor types, D1 dopaminergic receptor which is
expressed by MSNs projecting to the substantia nigra, and D2
dopaminergic receptor which is expressed by MSNs projecting to the globus
pallidus. Additionally, dopaminergic receptors have been shown to
suppress Ca2+ influx through L-type Cav channels in rodent
striated MSNs.

[0035]Calcium loading of neurons is an autonomous process. It is
contemplated that this process is potentially tied to the deafferentation
of neurons that leads to dopaminergic disorders like Parkinson's Disease.
It is contemplated that the deafferentation of neurons is a consequence
of altered modulation of the synaptically targeted Cav 1.3a calcium
channels by the D2 receptor and that partial disruption of these
channels prevents the structural adaptation following dopamine depletion.
Cav1.3a channels control synaptic plasticity and connectivity of
striatal MSNs. D2 receptor activation reduces Cav 1.3 channel
open probability whereas D1 receptor activation increases channel
open probability, or doesn't change it significantly. Genetic deletion of
Cav1.3a channels results in a dramatic increase in glutamatergic
synapsis and spines in MSNs suggesting that calcium flux through this
channel is a critical negative regulator of synapse stability. The loss
of dopamine receptors seen in PD leads to a loss of D2 receptors,
and consequently to an increase in the Cav 1.3 channel open
probability, followed by increases in intracellular calcium into spine
heads of stratopallidal neurons, and the eventual loss of glutamatergic
synapses in these neurons. The synaptic loss effectively causes
deafferentation of the striatopallidal neurons, preventing them from
fulfilling their role in motor control. This loss is contemplated to be a
major factor contributing to the pathophysiology underlying dopaminergic
disorders such as PD.

[0036]Disruption of Cav 1.3 channels prevents this deafferentation
following the loss of dopamine, and the targeting of Cav 1.3
channels does not depend upon retention of dopaminergic neurons nor is it
affected by alteration in signaling pathways triggered by disease
progression. For example, when a Cav 1.3 knock-out mouse was treated
with rotenone, a pesticide suggested to cause idiopathic PD, the SNc
neurons looked indistinguishable from untreated tissue whereas the
wild-type (wt) mice exhibited SNc deafferentation (FIG. 5). As will be
later demonstrated, these calcium channel knock-out mice demonstrate no
ill side affects due to the deletion based upon behavioral studies
(Example 5). The disruption of the Cav 1.3 calcium channels, either
through pharmacological blockade or channel deletion, lead to an altered
pacemaking mechanism where SNc dopaminergic neurons switch to sodium
channel dependent pacemakers without changing the pacemaking rate. The
altered pacemaking in dopaminergic neurons following Cav1.3 deletion
does not depend upon alterations in the sodium channel expression or
function, rather it depends upon modulation of endogenously expressed
hyperpolarization activated cation channels, which explains why normal
neurons are capable of compensating when disruption of Cav 1.3
calcium channels occurs (e.g. through pharmacological blockade).

[0037]Therefore, targeted pharmacological (e.g., antagonists, drugs,
agents, and the like) or genetic disruption (e.g., siRNA, and the like)
of Cav 1.3a is a novel therapeutic strategy that does not depend
upon an intact dopaminergic innervation of the striatum as do current
treatment strategies. It is contemplated that the prevention of
deafferentation and subsequent dopamine loss found in some dopaminergic
disorders will ameliorate the symptoms of the disease, and the specific
targeting of a cellular protein will significantly decrease the side
affects of current dopamine replacement strategies. The combination
therapy of disrupting the Cav 1.3a and the administration of
existing therapies (e.g., levodopa, carbidopa, etc.) will broaden the
window for treatment such that both cellular deafferentation will be
averted and dopamine levels will be ameliorated leading to a better
quality of life for those afflicted with dopaminergic disorders.

[0038]In one embodiment, the method of the invention comprises the
modulation of L-type calcium channels found in dopaminergic neurons. In
some embodiments, the modulation of L-type calcium channels involves
modulating Cav1.3 channels found in dopaminergic neurons. In some
embodiments, modulation of Cav1.3 channels further involves
modulating a subtype of the Cav1.3 channel, the Cav1.3a
channel. In some embodiments, the modulation of Cav1.3a channels
provides a treatment for Parkinson's Disease and other dopamineric
disorders of the basal ganglia (e.g. parkinsonian-type disorders, and the
like). In some embodiments, the present invention provides methods,
compositions, and kits for use in the modulation of Cav1.3a
channels. It is contemplated that Cav 1.3a channels may be modulated
using any methods including, but not limited to, biochemical, genetic,
and other methods known in the art.

[0039]Some embodiments of the present invention relate to therapeutic
methods and compositions for treating a subject having a dopaminergic
disorder or healthy subjects. In some embodiments, the method of
treatment comprises the administration of an antagonist, agent, compound,
or drug to a subject having a dopaminergic disorder or to healthy
subjects (e.g., prophylactic treatment), such as subjects with a
predisposition to, or risk of, acquiring a dopaminergic disorder (e.g.,
exposure to environmental toxins such as rotenone, genetic disposition,
etc.). In some embodiments, the antagonist physically interacts with the
Cav 1.3a calcium channel, or the antagonist blocks production of the
Cav 1.3a calcium channel, e.g. by inhibiting translation of the
receptor gene into a protein product. In one embodiment, the antagonist
is a siRNA that inhibits translation of the Cav 1.3a calcium channel
gene. Another embodiment comprises the administration of a calcium
channel blocker(s) to a subject having a dopaminergic disorder. One
embodiment comprises the administration of an antibody, antibody
fragment, or peptide that would block the calcium channel. A preferred
embodiment of a therapeutic method of treatment comprises the
administration of a calcium channel blocker from the dihydropyridine
class of compounds. For example, dihydropyridine calcium channel blockers
include, but are not limited to isopropyl 2-methoxyethyl
1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylate
(e.g., nimopidine, Nimotop® Bayer Corporation NDA#18-869),
3,5-pyridinedicarboxylic acid,
4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-, methyl 1-methylethyl
ester (e.g., isradipine, DynaCirc® Sandoz Pharmaceuticals
NDA#19-546), and dimethyl
1,4-hydro-2,6-dimethyl-4-(o-nitrophenyl)-3,5-pyridinecarboxylate (e.g.,
nifedipine Biovail Laboratories, Inc. ANDA#75-269, Adalat® Bayer
Corporation, U.S. Pat. Nos. 4,892,741 and 5,264,446, ELAN Pharmaceuticals
ANDA#75-128, Mylan Pharmaceuticals ANDA#75-108) and derivatives thereof
(all cited references are incorporated herein by reference). It is
further contemplated that dihydropyridine analogs are administered (e.g.,
nilvadipine, mesudipine, and the like). A preferred embodiment comprises
the administration of a dihydropyridine calcium channel blocker or analog
thereof to a subject suffering from a dopaminergic disorder. A preferred
embodiment comprises the administration of isradipine to a subject
suffering from a dopaminergic disorder. Typically, an effective amount of
a dihydropyridine calcium channel blocker can range from about 0.01 mg
per day to greater than 2000 mg per day for an adult, although other
doses are contemplated. Preferably, the dosage ranges from about 1 mg per
day to about 120 mg per day. More preferably the dosage ranges from about
20 mg per day to about 90 mg per day. It is contemplated that the dosage
is administered, for example, continuously on a daily, weekly, monthly,
or yearly basis. Dihydropyridine calcium channel blockers are well
tolerated by human subjects, and are used in therapeutic regimens for
other diseases and disorders (e.g., cardiovascular conditions, etc.).

[0040]In some embodiments the present invention provides methods of
storage and administration of the antagonist, agent, compound, or drug in
a suitable environment (e.g. buffer system, adjuvants, etc.) in order to
maintain the efficacy and potency of the agent, compound, or drug such
that its usefulness in a method of treatment of a dopaminergic disorder
is maximized. For example, protein agents, chemicals or nucleic acids
benefit from a storage environment free of proteinases and other enzymes
or compounds that could cause degradation of the protein, chemical, or
nucleic acid.

[0041]A preferred embodiment is contemplated where the antagonist, agent,
compound, or drug is administered to the individual as part of a
pharmaceutical or physiological composition for treating dopaminergic
disorders. Such a composition can comprise an antagonist and a
physiologically acceptable carrier. Pharmaceutical compositions for
co-therapy can further comprise one or more additional therapeutic
agents. The formulation of a pharmaceutical composition can vary
according to the route of administration selected (e.g., solution,
emulsion, capsule). Suitable pharmaceutical carriers can contain inert
ingredients that do not interact with the antagonist of Cav 1.3a
function and/or additional therapeutic agent. Standard pharmaceutical
formulation techniques can be employed, such as those described in
Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
Suitable physiological carriers for parenteral administration include,
for example, sterile water, physiological saline, bacteriostatic saline
(saline containing about 0.9% benzyl alcohol), phosphate-buffered saline,
Hank's solution, Ringer's-lactate and the like. Methods for encapsulating
compositions (such as in a coating of hard gelatin or cyclodextran) are
known in the art (Baker, et al, "Controlled Release of Biological Active
Agents", John Wiley and Sons, 1986). The particular co-therapeutic agent
selected for administration with an antagonist of Cav 1.3a calcium
channel will depend on the type and severity of the dopaminergic disorder
being treated as well as the characteristics of the individual, such as
general health, age, sex, body weight and tolerance to drugs.

[0042]In some embodiments the therapeutic agent is administered by any
suitable route, including, for example, orally (e.g., in capsules,
suspensions or tablets) or by parenteral administration. Parenteral
administration can include, for example, intramuscular, intravenous,
intraarticular, intrathecal, subcutaneous, or intraperitoneal
administration. The therapeutic agent (e.g., Cav 1.3a antagonist,
nucleic acid, additional therapeutic agent) can also be administered
transdermally, topically, by inhalation (e.g., intrabronchial,
intranasal, oral inhalation or intranasal drops) or rectally.
Administration can be local or systemic as indicated. The preferred mode
of administration can vary depending upon the particular agent chosen,
however, oral or parenteral administration is generally preferred. A
timed-release, subcutaneous mode of administration is also contemplated.
For example, a therapeutic agent is inserted under the skin either by
injection, and/or by placing a solid support that has been previously
impregnated or which contains (e.g., a capsule) the therapeutic agent,
under the skin. An effective amount of the therapeutic agent is then
released over time (e.g., days, weeks, months, and the like) such that
the subject is not required to have a therapeutic agent administered on a
daily basis. In some circumstances where high brain levels of the
antagonist are desired, intrathecal injection or direct administration
into the brain tissue is contemplated.

[0043]Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets or tablets, wherein each preferably contains a predetermined
amount of the active ingredient; as a powder or granules; as a solution
or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water
liquid emulsion or a water-in-oil liquid emulsion. In other embodiments,
the active ingredient is presented as a bolus, electuary, or paste, etc.

[0044]In some embodiments, tablets comprise at least one active ingredient
and optionally one or more accessory agents/carriers and are made by
compressing or molding the respective agents. In some embodiments,
compressed tablets are prepared by compressing in a suitable machine the
active ingredient in a free-flowing form such as a powder or granules,
optionally mixed with a binder (e.g., povidone, gelatin,
hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,
disintegrant (e.g., sodium starch glycolate, cross-linked povidone,
cross-linked sodium carboxymethyl cellulose) surface-active or dispersing
agent. Molded tablets are made by molding in a suitable machine a mixture
of the powdered compound (e.g., active ingredient) moistened with an
inert liquid diluent. Tablets may optionally be coated or scored and may
be formulated so as to provide slow or controlled release of the active
ingredient therein using, for example, hydroxypropylmethyl cellulose in
varying proportions to provide the desired release profile. Tablets may
optionally be provided with an enteric coating, to provide release in
parts of the gut other than the stomach.

[0045]Formulations suitable for parenteral administration include aqueous
and non-aqueous isotonic sterile injection solutions which may contain
antioxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include suspending
agents and thickening agents, and liposomes or other microparticulate
systems which are designed to target the compound to blood components or
one or more organs. In some embodiments, the formulations are
presented/formulated in unit-dose or multi-dose sealed containers, for
example, ampoules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile liquid
carrier, for example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from
sterile powders, granules and tablets of the kind previously described.

[0046]It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention may
include other agents conventional in the art having regard to the type of
formulation in question, for example, those suitable for oral
administration may include such further agents as sweeteners, thickeners
and flavoring agents. It also is intended that the agents, compositions
and methods of this invention be combined with other suitable
compositions and therapies. Still other formulations optionally include
food additives (suitable sweeteners, flavorings, colorings, etc.),
phytonutrients (e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.),
vitamins, and other acceptable compositions (e.g., conjugated linoelic
acid), extenders, and stabilizers, etc.

[0048]In other embodiments, the present invention provides methods of
screening compounds for their ability to inhibit Cav1.3a channels. In
some embodiments, the present invention provides drug-screening assays
(e.g., to screen for drugs effective in treating dopaminergic disorders).
For example, the present invention contemplates methods of screening for
compounds that modulate (e.g., decrease) the expression level or activity
of a Cav 1.3a calcium channel. In one embodiment, the expression
level of a Cav 1.3a calcium channel or its activity is detected in
vitro in a subject upon administration of a candidate compound. The
presence of a Cav 1.3a calcium channel or its continued or increased
activity is indicative of a candidate compound that is not preventing a
dopaminergic disorder. In some embodiments, the expression level or
activity of a Cav 1.3 a calcium channel is detected using an in
vitro assay, for example, an enzyme-linked immunosorbent assay, or other
assays which utilize a labeled (e.g., fluorescent, luminescent,
calorimetric, radioactive) compound for detection of a protein product or
channel activity. In other embodiments, the expression level of Cav
1.3a calcium channels can be detected using RT-PCR techniques as
described herein. Antagonists of Cav 1.3a calcium channels can be
identified, for example, by screening libraries or collections of
molecules, such as the Chemical Repository of the National Cancer
Institute, as described herein or using other suitable methods.
Antagonists thus identified find use in the therapeutic methods described
herein. Another source for identifying potential antagonists of Cav
1.3a calcium channels are combinatorial libraries, which can comprise
many structurally distinct molecular species. Combinatorial libraries can
be used to identify compounds or to optimize a previously identified
compound. Such libraries can be manufactured by well-known methods of
combinatorial chemistry and can be screened by suitable methods, such as
those described in Molecular Cloning: A Laboratory Manual Sambrook J et
al Eds, Cold Harbor Spring Laboratory Press.

[0049]In some embodiments, drug screening assays are performed in animals.
Any suitable animal may be used including, but not limited to, baboons,
rhesus or other monkeys, mice, or rats. Animal models of dopaminergic
disorders are generated, and the effects of candidate drugs on the
animals are measured. In preferred embodiments, dopaminergic disorders in
the animals are measured by detecting levels of Cav 1.3a calcium
channels in the affected tissues (e.g. SNc, MSN, other neuronal tissues)
of the animals. The expression level or activity of related Cav 1.3a
calcium channels may be detected using any suitable method, including,
but not limited to, those disclosed herein (e.g., tissue analysis,
nucleic acid analysis, behavioral analysis, etc.).

[0050]The present invention is not limited by the nature of the antagonist
used in the therapeutic or screening methods of the invention. In one
embodiment, the antagonist is a nucleic acid such as a small interfering
RNA (siRNA), which inhibits the translation of the mRNA encoding the
Cav 1.3a channel. Creation and use of siRNA is well known by those
skilled in the art. For example, specialized software such as
BLOCK-iT® RNAi Designer (Invitrogen Corporation) designs targeted RNAi
molecules to user defined sequences, and reference manuals (e.g., Hannon
G J ed., 2003, RNAi: A Guide to Gene Silencing, Cold Spring Harbor
Laboratory Press, p. 436.) to RNA interference applications are readily
available, and are incorporated by reference herein in their entirety.

[0051]In some embodiments, an antagonist of Cav 1.3a calcium channels
does not significantly inhibit the function of other neuronal calcium
channels (e.g., Cav 1.2, Cav 1.3b N-type, P/Q-type calcium
channels, and the like). Such Cav 1.3a-specific antagonists can be
identified by suitable methods, such as by suitable modification of the
methods described herein. For example, cells which do not express
Cav 1.3a but do express one or more other neuronal calcium channels
(e.g., Cav 1.2, Cav 1.3b N-type, P/Q-type calcium channels, and
the like) can be screen for channel specificity. Such cells or cellular
fractions (e.g., membranes) obtained from such cells can be used in a
suitable binding or activity assay. For example, if a cell lacks Cav
1.3a and contains only Cav 1.2, the Cav 1.3a antagonists can be
assayed for the capacity to inhibit expression or activity of the
Cav 1.2 calcium channel relative to the Cav 1.3a channel.

[0052]In another embodiment, the antagonist of a Cav 1.3a calcium
channel is an agent that inhibits a mammalian Cav 1.3a calcium
channel. Preferably, the antagonist of the Cav 1.3a calcium channel
is a compound that is, for example, a small organic molecule, natural
product, protein (e.g., antibody, peptide fragment), nucleic acid, or
peptidomimetic. Antagonists of Cav 1.3a calcium channels can be
prepared and/or identified using suitable methods, such as the methods
described herein or suitable modifications thereof.

[0053]The following examples are provided in order to demonstrate and
further illustrate certain preferred embodiments and aspects of the
present invention and are not to be construed as limiting the scope
thereof.

Example 1

Preparation of Experimental Mouse Brain Slices

[0054]Brain slices of mice were prepared for use in different experimental
procedures such as whole cell patch clamp and fluorescent staining
procedures. Slices were obtained from 17-25 day old C57BL/6 mice
(Harlan), Cav1.3.sup.-/- mice (were re-derived from mice obtained from
Joerg Striessnig) or BAC D1/BAC D2 EGFP transgenic mice (obtained from
Nathaniel Heintz), and P16-17 rats. All animals were handled in accord
with Northwestern University ACUC and NIH guidelines. Coronal slices
containing the striatum or mesencephalon were prepared at a thickness of
300-350 μm. Mice were prepared and sacrificed in one of two ways; 1)
the mice were anesthetized deeply with ketamine and xylaxine,
transcardially perfused with oxygenated, ice-cold, artificial cerebral
spinal fluid (ACSF) and decapitated, or 2) the mice or rats were deeply
anesthetized with halothane and decapitated without perfusion. The brains
were rapidly removed and sectioned in oxygenated, ice-cold, ACSF using a
Leica VT1000S vibratome (Leica Microsystems). When the mice were prepared
and sacrificed using ketamine, the ACSF sectioning solution contained 124
mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 26 mM NaHCO3,
1.2 mM NaH2PO4, and 10 mM D-(+)-glucose, while halothane
prepared mice ACSF sectioning solution contained 194 mM sucrose, 30 mM
NaCl, 4.5 mM KCl, 1 mM MgCl2, 26 mM NaHCO3, 1.2 mM
NaH2PO4 and 10 mM D-glucose. The solutions were periodically
checked and adjusted to ensure the osmolarity stayed near 300 mOsm/l.
Once sliced, the brain sections were transferred to a holding chamber
where they were completely submerged in ACSF bubbled continuously with
95% O2 and 5% CO2 and maintained at room temperature
(22°-23° C.) for at least 1 hour before using.

Example 2

Electrophysiology Procedures

[0055]Whole cell patch clamp techniques were used on tissue slices to
evaluate the effect of experimental conditions on the voltage gated
calcium channels. Whole cell voltage-clamp or current-clamp recordings
were performed using standard techniques (Choi S and Lovinger D M, 1997,
Proc. Natl. Acad. Sci. 94:2665-2670; Day M et al., 2005, J. Neurosci.
25:8776-87). Individual slices were transferred to a submersion style
recording chamber and continuously superfused with ACSF at a rate of 2-3
ml/min at 31-33° C. Whole cell voltage and current clamp
recordings were performed on dopaminergic neurons detected in the slice
with the help of an infrared-differential interference contrast (IR-DIC)
video microscope upon which was mounted an Olympus OLY-150
camera/controller system (Olympus, Japan). For all experiments,
experimental drugs were superfused with a temperature controlled system.
Isradipine, nimodipine, and tetrodotoxin were made up as stock solutions
and diluted immediately before use. Patch electrodes were made by pulling
TW150F-4 (World Precisions Instruments, Sarasota Fla.) or BF150-86-10
glass on a P-97 Flaming/Brown micropipette puller (Sutter Instrument Co.)
and fire polished before recording. Pipette resistance was typically
2.5-6 MW after filling with internal solution.

[0056]L-type calcium channels can be inhibited by dihydropyridine
antagonists, such as nimodipine (FIG. 1) and isradipine (FIG. 4).
Blockade with isradipine activates a homeostatic mechanism which involves
adenylyl cyclase and hyperpolarization activated cation channels, that
restores pacemaking and dopaminergic neuron cell function through an
altered pacemaking mechanism. The sustained blockade of Cav1.3
channels leads to re-emergence of the altered pacemaking mechanism that
is dependent upon sodium and hyperpolarization activated cation channels.
This shows that blockade of Cav1.3 channels does not have
significant side effects on brain dopaminergic function nor are obvious
behavioral consequences observed. As well, blockade of tetrodotoxin (TTX)
sensitive sodium channels eliminates spikes but not the underlying
oscillations that drive pacemaking.

Example 3

Procedure for Two Photon Laser Scanning Microscopy

[0057]Two photon laser scanning microscopy (2PLSM) was performed on mouse
tissue samples to visualize intracellular conditions. 2PLSM images of
medium spiny neurons in 275 μm thick corticostriatal slices were
visualized with Alexa Fluor 594 (50 μM) by filling through the patch
pipette. Following break in, the dye was loaded for at least 15 minutes
prior to imaging. 2PLSM red signals (580-640 nm) were acquired using 810
nm excitation with 90 MHz pulse repetition frequency and ˜250 fs
pulse duration at the sample plane. Maximum projection images of the soma
and dendritic field were acquired with a 60X/0.9NA water-dipping lens
with 0.27 μm2 pixels and 2.6 μs pixel dwell time; ˜80
images were taken using 0.7 μm focal increments. High magnification
projections of dendritic segments taken 50-100 μm from the soma were
acquired with 0.17 μm2 pixels and 10.2 μs dwell time and
consisted of ˜20 images taken at 0.5 μm focal steps. 2PLSM green
signals (500-550 nm) were acquired from neurons using 810 nm excitation.

[0058]The two-photon excitation source was a Chameleon-XR tunable laser
system (705 nm to 980 nm) utilizing Ti:sapphire gain medium with
all-solid-state active components and a computer optimized algorithm to
ensure reproducible excitation wavelength, average power, and peak power
(Coherent Laser Group). Excitation at 810 nm with 90 MHz pulse repetition
frequency and ˜250 fs pulse duration at the sample plane was used
for the two-photon excitation. Laser average power attenuation was
achieved with two Pockels cell electro-optic modulators (models 350-80
and 350-50, Con Optics). The two cells were aligned in series to provide
enhanced modulation range for fine control of the excitation dose (0.1%
steps over four decades). Laser scanned images were acquired with a
Bio-Rad Radiance MPD system (Hemel Hempstead). Fluorescence emission was
collected by external or non-de-scanned photomultiplier tubes (PMT's).
Green fluorescence (500 to 550 nm) was detected by a bialkali-cathode PMT
and red Once fluorescence emission was collected, the system digitized
the current from detected photons to 12 bits. The laser light transmitted
through the sample was collected by the condenser lens and sent to
another PMT to provide a bright-field transmission image in registration
with the fluorescent images. The stimulation, display, and analysis
software was a custom-written shareware package (WinFluor and
PicViewer-John Dempster, Strathclyde University, Glasgow, Scotland; UK).

[0059]As can be seen in FIG. 3, pacemaking is accompanied by large
fluctuations in calcium levels in dendrites of SNc dopaminergic neurons
thereby demonstrating that calcium levels are important in activity of
these cells.

Example 4

Single Cell Reverse Transcription Polymerase Chain Reaction Procedure

[0060]Single cell reverse transcription polymerase chain reaction
(scRT-PCR) was performed on cell nucleic acids to determine the levels of
specific mRNA that a particular cell was producing. Neurons were acutely
isolated, harvested and profiled using protocols similar to those
previously described (Tkatch T et al., 2000, J. Neurosci 20:579-88).

[0061]As demonstrated in FIG. 2, the long splice variant of the
Cav1.3 gene, Cav1.3a, is present in SNc dopaminergic neurons
and medium spiny neurons.

Example 5

Behavioral Analysis Techniques Used to Evaluate Cav1.3 Knock-Out Mice

[0062]Behavioral analysis was performed on Cav1.3 knock-out mice and
compared with the behavioral performance of wt mice in order to determine
if the deletion of the Cav1.3 subunits affected mouse behavioral
phenotypes. Mice were evaluated using several different techniques; the
pole test, the elevated plus maze (EPM), the cross maze, and a swimming
version of the cross maze. Motor agility was assayed using the pole test,
anxiety related behavior and cognitive/learning behaviors were evaluated
using the cross maze, the swimming cross maze, and the EPM.

[0063]Test subjects were two cohorts of male mice (N=8 & 11). Each cohort
contained both Cav1.3 knock-out mice (N=9) and wt littermates
(N=10). All of the mice were group housed. The mice were placed on a
water restricted diet prior to cross maze testing so that they would be
thirsty and work for water rewards. The mice received water during
testing and for one hour at the end of the day when they had one hour of
free access to water in their home cage. The weight, appearance and
behavior of the mice were monitored throughout the experiment in order to
insure that the mice were in healthy condition (all procedures were
approved by Northwestern University's ACUC committee). The experimenter
was blind as to the genotype of the mice during data collection.

[0064]The pole test (Matsuura K, et al., 1997, J. Neurosci. Meth. 73:45-8)
was used to assess gross motor agility. Mice were placed at the top of an
8 mm diameter pole with their head pointed up. The time taken for them to
descend the 55 cm to the bottom of the pole was recorded. Results
indicate that there was no significant difference between wt and
knock-out mice for the time it took to invert themselves and descend the
pole to the bottom.

[0065]The land based cross-maze was used to test the mice for motor and
cognitive/learning abilities. The maze consisted of an elevated cross
shaped platform with four white arms of equal length (35×6.5 cm)
that extended from a central area (6.5×6.5 cm) and which were
enclosed by clear Plexiglas walls (15 cm tall). The maze was based on
that described by Middei S, et al., 2004, Behav. Brain Res. 154:527-34,
except that the sidewalls extended the entire length of the maze, and the
end of each arm had a depression to contain a water reward (a 25 μl
drop). Subjects received five consecutive days of habituation sessions.
During these sessions, the mice were introduced into the maze at the
south end, and the east and west arms of the maze were blocked by
Plexiglas barriers in order not to induce a bias towards a particular
east or west arm. The depression well at the end of the north arm
contained 25 μl of water. Each mouse was given five trials per session
with the opportunity to explore the maze, discover the water in the
depression at the end of the north arm, and learn to drink the water. The
mice were removed from the maze 15 seconds after drinking. Mice that did
not find the water within two minutes were guided to the water and then
removed 15 seconds after drinking.

[0066]Experimental sessions began twice a day at the conclusion of the
habituation sessions. Mice were typically started from the distal end of
the south arm and access to the north arm of the maze was blocked by a
Plexiglas barrier so that the maze became a "T" maze. The maze was kept
in a room with the same visual cues in place during each session. The
mice were given five trials in which the north arm of the maze was
blocked. The goal arm (the east arm) contained 25 μl of water while
the west arm contained no water. The mice were introduced via the south
arm and allowed to choose an arm. Once the mice picked an arm, they were
enclosed in the arm for 15 seconds that was enough time for the mouse to
reach the end of the arm, drink the water (if the correct arm was chosen)
and scan the surroundings. No correction methods were implemented for
incorrect arm choice. A mouse was considered to have reached the center
of the maze when its front paws crossed the border between the south arm
and center region. The mice frequently explored the east or west arms
with their nose and front paws, but an entry was considered to have been
made only after both hind paws and the snout crossed the border of the
center region and a side arm.

[0067]Movement speed was calculated for traversing the cross maze. Mice
typically went to the center area very quickly. The trial with the
shortest latency for each session was analyzed using a repeated measures
ANOVA. The results indicate no significant difference between the wt and
knock-out mice. The mice spent much of their time in the center portion
of the cross maze which suggested that they might be exhibiting anxiety
related behaviors. The number of fecal boli deposited by each mouse on
the fifth day of training was recorded and analyzed for a difference
between groups with a t-test. No significant difference was revealed.

[0068]A more rigorous examination of anxiety and exploration was performed
by testing the mice on the EPM (VideoMot2, TSE, Midland, Mich.) for a
single five minute session. The software calculated the time spent by
each mouse in the open and closed arms, as well as the number of entries
into each type of arm. None of the variables measured exhibited a
significant difference between the wt and knock-out mice. The movement
speed during exploration in the EPM was measured and no significant
difference was found between genotypes.

[0069]Cognitive abilities were additionally assessed using the cross maze.
A repeated measures ANOVA of genotype by session for the percent of
correct choices indicated no significant difference between genotypes,
but a significant increase from 49.5% correct on session 1 (50% indicates
choosing randomly) to 83.2% correct on session 5 (ANOVA, F 4, 68=14.1,
P<0.0001).

[0070]As mentioned earlier, the mice in the cross maze quickly ran to the
center and then often stayed in the center or traversed the south start
arm. The latency between entering the center area and finally choosing a
side arm to enter was analyzed using a repeated measures ANOVA for the 12
sessions, and genotype as a factor. The results indicated no significant
difference in this choice latency between the wt and knock-out mice. A
detailed analysis of the exploratory activity prior to committing an
entry to the east or west arm was done towards the end of training. Those
results indicated no significant difference between genotypes in terms of
how often the mice explored the east or west arm with nose pokes, but
there was a significant preference (13.4 versus 5.5 counts) for exploring
the east, rewarded arm (F 1,9=22.0, P=0.001) when the two genotypes were
considered together.

[0071]A probe trial was conducted after the initial five trials were
completed. Mice were started from the far end of the north arm for this
trial and access to the south arm was blocked. The east and west arms
both contained 25 μl of water, and the subjects were allowed to pick
and explore an arm. The subjects were given a maximum of two minutes to
choose an arm during all trials. If this time limit was exceeded, the
subject was removed from the maze and given another trial. In order to
remove any olfactory cue, the maze was wiped down with a sponge damped
with water after each trial. Data were averaged for the two sessions per
day prior to analysis. An analysis of the probe trials suggests that
knock-out mice were choosing which side to enter in a random fashion
while the wt mice chose in a manner indicating a significant preference
for hippocampally based spatial behavior rather than a striatal based
motor response (F 1, 17=4.5, p=0.049), i.e., the wt mice continued to
prefer the east (rewarded) arm even when started from the north instead
of the south arm. This difference between the knock-out and wt mice
appeared to be dominated by the last session on day five since it was the
only session to show a significant difference between the groups when
analyzed with individual t-tests (t=2.1, df=17, p=0.049). According to
test results, the knock-out mice were responding in a random fashion and
the wt mice were exhibiting a preference for a spatially based behavior
which is expected for C57Bl/6 mice (Middei S, et al., 2004, Behav. Brain
Res. 154:527-34). These data could be interpreted to mean that wt C57BL6
mice are dominated by hippocampal based spatial behavior and that the
striatal system of Cav1.3 knock-out mice are more able to influence
motor behavior.

[0072]The behavioral analysis of the land based cross maze was complicated
by the tendency of the mice to stop ambulating during a trial. Therefore,
a water based version was created and used to determine if any
differences between genotypes were present. The mice swam well and did
not stop and float during the test. The maze was made of clear acrylic
and had arms that extended 77.5 cm from one end to the other. The alley
ways were 15 cm wide with 17.5 cm high walls. The escape platform was
6×6 cm and was elevated 10.5 cm from the base of the maze. There
were no edges for the mouse to grab and the alleys were wide enough that
the mouse could not brace itself between the walls. The maze was
submerged within a pool of 25° C. water that was made opaque with
white tempera paint. HVS Image software was used to collect latency and
path length data. A repeated measures ANOVA for the percent of correct
choices across 10 training sessions indicated significant improvement in
choosing the correct arm with no significant difference between genotype,
and no significant interaction of group and learning. The percent of
correct responses increased from a minimum of 60% correct on the second
session (50% is random) to a maximum of 100% correct on the eighth
session. The mean latency to find the hidden escape platform in the
rewarded arm of the maze was also measured and analyzed. This escape
latency decreased significantly during the first five sessions from a
maximum of 13.5 seconds on session two to a minimum of 4.0 seconds on
session five. There was no significant difference between the wt and
knock-out mice, and no interaction of group and training. The same
results were found when the distance traveled to the goal was analyzed.

[0073]The mice were put back into the water cross maze two days later and
released to swim to a visible platform (instead of a hidden platform)
that was located in the arm opposite that which had been rewarded with
the hidden platform. This test is considered sensitive to striatal
involvement since striatal based habit learning might impair the ability
of the mouse to execute a new response pattern. The results indicated
improvement during the first session, but there was no significant
difference between the wt and knock-out mice for the latency to climb
onto the escape platform during any of the three days of testing. The
first trial of the visible testing is interesting and suggests that the
knock-out mice have more "cognitive flexibility" than the wt mice (i.e.
the hippocampus can overcome the striatum). Finally, the latency for the
mice to reach the escape platform in the water cross maze during the last
five days of testing was analyzed with a repeated measures ANOVA. The
results indicated no significant difference between the groups.

[0074]As described above, when Cav1.3 knock-out mice were compared
with litter mate controls for differences in motor behavior, agility,
anxiety, learning, and preference for spatial or response based
ambulation no differences due to genotype were noted, except that there
was a hint of a preference for spatially guided ambulation in wt mice and
a random probability that a knock-out mouse would use either spatial
guidance or response guidance. Therefore, the elimination of the
Cav1.3 calcium channel, thereby shifting the pacemaking mechanism to
the more juvenile form of pacemaking that shifts dopaminergic neurons to
sodium channel dependent pacemaking, does not lead to any obvious
behavioral consequences.

[0075]Another way of inhibiting Cav 1.3 channels in SNc dopaminergic
neurons is to inhibit translation of the Cav 1.3 mRNA using siRNA
technique. The target sequences for the siRNAs are selected to avoid
potential inhibition of other calcium channels. For example, conserved
regions, like transmembrane domains and the proline rich region, are
avoided. The greatest sequence diversity in calcium channel subunits is
found in the 3' region of the mRNA that codes for the cytoplasmic carboxy
terminal region. This segment of the mRNA is preferably targeted. BLAST
sequence analysis programs can be used to screen candidate siRNA for
specificity. Efficiency and specificity of the calcium channel inhibition
can be assessed using real-time quantitative PCR and Western blotting. In
some embodiments, viral constructs that express small hairpin RNA (shRNA)
are used for administration.

[0076]All publications and patents mentioned in the above specification
are herein incorporated by reference. Various modifications and
variations of the described method and system of the invention will be
apparent to those skilled in the art without departing from the scope and
spirit of the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be understood
that the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the described
modes for carrying out the invention that are obvious to those skilled in
the relevant fields are intended to be within the scope of the following
claims.